ABSTRACT (PROJECT 3) The molecular mechanisms underlying cellular vulnerability in Alzheimer?s disease are unclear. The objective of this project is to clarify the molecular mechanisms responsible for select cell vulnerability and pathogenesis by applying spatial methods for highly multiplexed RNA and protein analysis in Alzheimer?s-affected tissue sections. We will optimize and standardize platforms for multiplexed fluorescence in situ hybridization (mFISH) to examine AD-linked gene sets derived from single nucleus transcriptome analysis, as well as immunohistochemistry (IHC) to examine pathological protein markers in the context of cell type populations in intact human brain tissue. These methods will then be applied to Alzheimer?s disease tissues from brain regions affected in different levels of disease severity to understand 1) the spatial organization of transcriptomically-defined cell types in these brain regions and changes in this organization with disease severity (e.g. selective vulnerability or proliferation), 2) molecular pathways disrupted in Alzheimer?s disease in the context of specific cell types, and 3) the relationship between these affected cell types and the markers of Alzheimer?s pathology, such as amyloid precursor protein and hyperphosphorylated tau. These data generated by mFISH and IHC together will provide a high-resolution map of the cellular and molecular consequences of clinically typical Alzheimer?s disease and an enhanced view of Alzheimer?s pathology, as well as a powerful technology platform for future analyses of larger and more varied cohorts.